Refolding Record:
| Protein | |
|---|---|
| Protein Name | Circumsporozoite protein |
| Abbreviated Name | CS |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Plasmodium falciparum |
| UniProt Accession | P02893 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 397 |
| Molecular Weight | 42596.3 |
| Pi | 5.01 |
| Molecular Weight | 42596.3 |
| Disulphides | Unknown |
| Full Sequence |
EALFQEYQCYGSSSNTRVLNELNYDNAGTNLYNELEMNYYGKQENWYSLKKNSRSLGENDDGNNNNGDNGREGKDEDKRDGNNEDNEKLRKPKHKKLKQPGDGNPDPNANPNVDPNANPNVDPNANPNVDPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNVDPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNKNNQGNGQGHNMPNDPNRNVDENANANNAVKNNNNEEPSDKHIEQYLKKIKNSISTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYENDIEKKICKMEKCSSVFNVVNSSIGLIMVLSFLFLN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Kolodny N, Kitov S, Vassell MA, Miller VL, Ware LA, Fegeding K, De La Vega P, Sacci JB Jr, Lanar DE. (2001) J Chromatogr B Biomed Sci Appl., 762, 77-86 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BLR(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 1 h |
| Expression Vector | pET32a |
| Expression Protocol | For expression of the PfCS protein, the plasmid was transformed in to BLR(DE3) cells. Using a Bioflo 3000 10l fermenter (NewBrunswick, Edison, NJ, USA) cells were grown at 37Cin SB medium (12 g/l tryptone, 24 g/l yeast extract, 6.3 ml/l glycerol, 12.5 g/l KHPO, 3.8g/l KHPO; pH7.2) with kanamycin to an optical densityat 600 nm (OD600) of 6.0. The PfCS protein production was induced by the addition of IPTG (isopropyl-b-D-thiogalac- topyranoside) to a final concentration of 0.2mM and cell growth continued for 1 h. At this point the OD 600 was 10 and cell growth had ceased. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 6.0 = 600 |
| Cell Disruption Method | Microfluidizer |
| Lytic Agent | Other |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | n/a |
| Solubilization Buffer | 6 M Gu-HCl |
| Refolding Buffer | 50 mM imidazole fraction |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 0.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Frozen denatured cell lysate (100 ml of lysate from 10 g wet paste) was thawed and loaded (2 ml/min) on to the Ni–NTA column through the 600E manifold using a 60-ml syringe. After loading, non specific and unbound proteins were removed by washing the resin with buffer N3 until the OD280 reached a back ground level. During this wash step the protein denaturant, 6MGu-HCl, was exchanged for 8 M urea. There folding–renaturation of the PfCS protein was then performed while bound on the Ni–NTA column using two linear gradients. The first gradient reduced the ure a concentration from 8 to 4 Min 30 min. This was followed with the second gradient that further reduced the urea concentration from 4 to 0 Min 3 h. This was followed by elution ofa major protein peak with buffer N5. Collection of the protein started when the OD went above the initial base line of 0.01 and finished when a new baseline was reached, higher than the starting base line due to the presence of the imidazolein the elution buffer. The protein sample eluted from the Ni–NTA column could bestoredat 48C after adding EDTA to a final 1 mM concentration. |
| Refolding Assay | Western Blot |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | refolding pH is not stated |